We have developed technology that enables rapid, comprehensive and high-throughput analysis of humoral immune responses to infectious disease antigens that is being used for subunit vaccine antigen discovery and to develop serodiagnostic tests. The method is based on printing microarray chips with proteins encoded by infectious microorganisms which cause medically important diseases in humans. Currently, we have expressed and printed more than 14,000 individual proteins from 6 bacteria, 17 viruses, and 2 parasites. The chips are probed with serum from infected patients and healthy controls to identify the dominant antibody responses, to determine the immunodominant antigen profile and identify a set of 10-20 serodiagnostic antigens for each infectious agent. By using a multiplicity of antigens for each infectious agent, serodiagnostic tests with statistically improved sensitivity and specificity can be configured. Here we will fabricate whole proteome microarrays for Coxiella burnetii, Rickettsia prowazekii, Brucella melitensis, Salmonella typhi, and non-homologous proteins from related strains of each agent. The arrays will be probed with serum from well-characterized infected subjects and healthy controls to identify the immunodominant and serodiagnostic antigen sets. We will also fabricate a chip containing 190 proteins from 16 arboviruses and 8 hantaviruses. The ability of this virus chip to distinguish well-characterized sera collected from humans and animals infected with the different viruses will be determined and the results compared with established assays done on the same samples. Deliverables generated from this project include plasmids, customized protein microarrays in a modular multiplex format, purified recombinant proteins and lateral flow (dipstick)/immunostrips devices, and will be accessible from the PSWRCE Protein Microarray Core on a recharge basis. These will be available to RCE investigators, government agencies and to private businesses through licensing agreements. The Protein Microarray Core will also provide chip probing and analysis services. Serodiagnostic antigen sets will be available for licensing to businesses interested in developing serodiagnostic tests. The immunodominant antigen sets, which may contain subunit vaccine antigens, will be available to commercial vaccine developers through licensing agreements. Multiplex serodiagnostic chips will be useful for determining whether military personnel or civilians, who enter a region where these agents are endemic, are exposed to any of them. They will also benefit public health monitoring activities to track annual changes in the prevalence of infections in endemic regions, and for monitoring the blood supply in developing countries. The chips will also be useful for assessing the spread of exposure in a population following a bioterrorism attack.